Abstract The completion of Chinese cabbage whole genome and the reduced cost of re-sequencing provide possibility for high-throughput development of InDel markers. In the present study, two Chinese cabbage inbred lines He102 and 06-247 with significant differences in phenotype such as bolting tolerance, virus resistance and tip-burn tolerance were re-sequenced for genome-wide InDels identification. The sequencing depth of these two lines was 10×and 8×respectively. A total of 330 218 polymorphic InDels loci were predicted based on the sequence data. The frequency of InDels occurrence was 1.2/kb, among which 11 238 loci were located in deduced coding region and represented for 5184 genes. We randomly selected 933 predicted polymorphic InDels loci that distributed on the 10 chromosomes of He102 and 06-247 respectively for PCR validation. Results showed that the positive rate varied greatly with sequencing depth. When the choosed loci from a higher sequencing depth material He102, the false positive rate was higher either, and vise versa. In the present study, the average false positive rate was 51.9% and 22.5% for the selected loci from He102 and 06-247, respectively. In other words, higher false positive rate based on higher-depth material reflected that the false positive loci were missed in lower-depth material; contrarily, lower false positive rate based on lower-depth material reflected that the positive loci were detected in higher-depth material. A total of 593 verified loci including 375 loci located in coding sequence (CDS), were identified. More than 90% of the polymorphism loci were co-dominant while about 5% of them were dominant. The rest couldn't produce clear band or multiple bands were generated. The detected polymorphic loci were not evenly distributed on ten chromosomes. Chromosome A01 contained the most number (86) of loci, while A08 contains only 29 loci. The co-dominant polymorphic loci were used to detect the residual heterozygous line of F7 which was generated by single seed descend from F2 of He102×06-247. The results showed that in F7 population, more than 90% of the alleles were homogenous while less than 10% of the them were remain heterogenous. The hemogenous/heterogenous state and the physical position of each locus in all 217 lines of F7 were confirmed. The residual heterozygous lines and the identified polymorphic markers provided useful information for elaborate decompose and fine mapping of QTLs/genes related to bolting, virus resistance and tip-burn tolerance in Chinese cabbage.
ZHANG Zhi-Gang,DIAO Zhi-Zhong,LI Qiao-Yun, et al. Development of InDels Markers and Their Usage in Detection of Residual Heterozygous Lines in Chinese Cabbage (Brassica rapa ssp. pekinensis) [J]. , 2016, 24(4): 510-518.
[1]Ayahiko S, Takeshi I, Kaworu E, et al..Deletion in a gene associated with grain size increased yields during rice domestication. [J].Nature genetics., 2008, 40:1023-1028.
[2]Konishi S, Izawa T, Lin SY, Ebana K, Fukuta Y, Sasaki T, and Yano M.An SNP caused loss of seed shattering during rice domestication[J].Science, 2006, 312(5778):1392-1396
[3]Wang X, Wang H, Wang J, et al.The genome of the mesopolyploid crop species Brassica rapa[J].Nature genetics, 2011, 43:1035-1039
[4]Yano M, Katayose Y, Ashikari M, et al..Hd1, a major photoperiod sensitivity quantitative trait locus in rice, is closely related to the arabidopsis flowering time gene CONSTANS[J].the Plant Cell, 2000, 12:2473-2484
[5]杜景红, 樊叶杨, 王磊，等.应用剩余杂合体衍生的近等基因系分解水稻产量性状[J].中国水稻科学, 2008, 22(1):1-7
[6]樊叶杨, 程式华, 范方军, 等.水稻第染色体短臂每穗实粒数和每穗颖花数的精细定位[J].核农学报, 2010, 24(6):1105-1109
[7]冯芳君.2006. SSR和InDel标记在水稻籼粳亚种分化与杂种纯度鉴定中的应用[D]. 硕士学位论文, 华中农业大学园艺学院, 导师: 罗利军, pp.22-35(Feng FJ. 2006. The study on Indica-Japanica differentiation and testing the seeds purity of hybrid rice by SSRs and InDels[D]. Thesis for M.S., College of Horticulture Huazhong Agricultural University. Supervisor:Luo L J, pp.22-35)
[8]惠麦侠.2011. 白菜叶缘裂刻基因的精细定位及BcCUC基因克隆和功能分析[D]. 硕士学位论文, 西北农林科技大学园艺学院, 导师: 张鲁刚, pp.18-20(Hui M X. 2011. Fine mapping of lobe-leafed gene and cloning and functional analysis of bounary-speicific BcCUC1 and BcCUC3 genes from non-heading Chinese cabbage (Brassica campestris ssp.chinensis) [D], Thesis for M.S., College of Horticulture, Northwest A&F University, Supervisor:Zhang LG, pp.18-20)
[9]李斯更, 沈镝, 刘博, 等.基于黄瓜基因组重测序的标记开发及其应用[J].植物遗传资源学报, 2013, 14(2):278-283
[10]林文春, 余守武, 阮关海, 等.水稻千粒重 基因的精细定位[J].核农学报, 2014, 28(2):217-223
[11]刘栓桃，张志刚，李巧云，等.光敏色素基因启动子突变及与大白菜开花时间的关联分析[J].农业生物技术学报, 2014, 22(7):853-861
[12]罗炬, 邵高能, 魏祥进, 等.一个控制水稻株高 的遗传分析[J].中国水稻科学, 2012, 26(4):417-422
[13]孟霖, 刘博, 林良斌, 等.白菜型油菜和菜薹的标记开发及其 群体遗传连锁图谱的构建[J].园艺学报, 2012, 39(8):1491-1500
[14]南海洋, 李英慧, 常汝镇, 等.基于大豆胞囊线虫病抗性候选基因的标记开发与鉴定[J].作物学报, 2009, 35(7):1236-1243
[15]邵高能, 唐绍清, 罗炬, 等.水稻剑叶形态与稻米粒形分析及相应剩余杂合体衍生群体的构建[J].分子植物育种, 2009, 7(1):16-22
[16]申璐, 沈火林, 柴敏, 等.采用和标记分析番茄品种基因组多态性[J].中国农业大学学报, 2011, 16(2):34-42
[17]唐棣，王志民.检测方法研究进展[J].上海交通大学学报农业科学版, 2007, 25(2):405-413
[18]王万兴.2013. 结球甘蓝高密度遗传连锁图谱的构建与主要农艺性状的QTL定位[D]. 博士学位论文，中国农业科学院蔬菜花卉研究所, 导师: 杨玉梅, pp.18-33(Wang WX. 2013. Construction of high-density Genetic linkage Map and QTL analysis for main agronomic traits in cabbage (Brassica oleracea L.var.capitata)[D], Dissertation for Ph.D., the Institute of Vegetables and Followers Chinese Academy of Agricultural Sciences, Supervisor: Liu YM, pp.18-33)
[19]吴为人, 李维明, 卢浩然.建立一个重组自交系群体所需的自交代数[J].福建农业大学学报, 1997, 26(2):129-132
[20]薛银鸽, 原玉香, 张晓伟, 等.利用标记鉴定大白菜杂交种豫新四号种子纯度[J].农业生物技术学报, 2014, 22(4):449-456
[21]张圣平, 苗晗, 程周超, 等.黄瓜果实苦味基因的插入缺失标记[J].农业生物技术学报, 2011, 19(4):649-653
[22]张曦.2013. 大白菜蜡粉基因的精细定位及表达分析[D]. 硕士学位论文，沈阳农业大学园艺学院，导师：冯辉, pp. 46-49(Zhang X. 2013. Fine mapping and gene expression of wax gene in Chinese cabbage (Brassica rapa L. ssp. pekinensis)[D]. Dissertation for Ph.D., College of Horticulture, Shenyang Agricultural University, Supervisor: Feng H. pp. 46-49)
[23]赵卓.2014. 大白菜抗根肿病基因CRb的精细定位, 硕士学位论文[D].沈阳农业大学园艺学院, 导师: 朴钟云, pp. 29-34(Zhao Z. 2014. Fine genetic and physical mapping of the CRb gene conferring resistance to clubroot disease in Brassica rapa[D]. Thesis for M.S., College of Horticulture, Shenyang Agricultural University, Supervisor: Piao Z Y, pp. 29-34)
